1. Field of the Invention
The present invention relates generally to an assay for simultaneously determining the presence of T cells and B cells in a biological specimen, and more particularly, to an immunoassay for determining the relative amounts of T and B cells and subpopulations thereof by specifically labeling said cells with differentiable particles and counting the particles under a microscope.
In vertebrates, including man, T and B cells form the two primary lymphocyte populations. Upon stimulation with antigen, B cells differentiate into plasma cells which secrete antibodies specific to the antigen. The role of T cells is less well defined, although it is clear that they mediate cellular immune responses such as delayed hypersensitivity, immune surveillance and graft rejection. Certain T cell subpopulations also play a role in regulating the B cell response to antigens.
In normal individuals, the lymphocyte population in the peripheral blood comprises from about 70 to 80 percent T cells and from about 10 to 20 percent B cells with the remaining cells referred to as "null" cells. The determination of the relative number of T and B cells is clinically important in diagnosing a wide variety of immunologically-mediated diseases, including lymphomas, lymphocytic leukemias and various immuno-deficiency diseases, such as congenital sex-linked agammaglobulinemia. The determination is also useful in evaluating immunocompetance and mechanisms of tissue damage in autoimmune diseases, such as systemic lupus erythematosus.
The T cell and B cell populations may further be divided into subpopulations distinguished by the presence of characteristic antigens on the cell surface membrane. The primary subpopulations of T cells are the helper (T.sub.H) and suppressor (T.sub.S) cells which function in humoral immune response. The primary B cell subpopulations correspond to the classes of immunoglobulin (Ig) present on the cell surface and produced by the plasma cell after stimulation. Determination of the relative amounts of the subpopulations of both B cells and T cells also promises to have clinical importance.
It is thus useful and desirable to provide a convenient and accurate method for determining the relative amounts of T cells and B cells and subpopulations thereof in a biological sample, such as human blood.
2. Description of the Prior Art
Heretofore, human B cells have been identified based on the presence of membrane-bound immunoglobulin. Typically, labeled anti-(human immunoglobulin) antibody is used to separate or otherwise identify the B cell population. The use of polyclonal anti-(human immunoglobulin) antibody coupled to polyacrylamide beads is discussed in Jabs, et al., "Identifying Human Lymphocyte Subpopulations," Lab. Manag., (January, 1980). Such beads are available from Bio-Rad Laboratories, Richmond, Calif. Enzyme-labeled goat anti-(human immunoglobulin) antibody is described in Technical Bulletin No. 95, Sigma Chemical Company, St. Louis, Mo.
T cells have been conventionally identified by their ability to spontaneously bind with sheep red blood cells to form "rosettes" visible under an ordinary light microscope. A description of this technique is found in Hudson, et al, "Practical Immunology," Blackwell Scientific Publications, Oxford, pp. 301-302 (1980).
Commercial kits are presently available for the assay of the B cell and T cell populations in human blood. One such kit, available from Kallestad Laboratories, Inc., Austin, Tex., utilizes labeled anti-(human immunoglobulin) antibody for detecting B cells and sheep red blood cells for identifing T cells in separate steps. Another kit, identified as the "T & B Cell Reagent Set," manufactured by Hybritech, Inc., La Jolla, Calif., combines labeled anti-T cell antibody (monoclonal) and labeled anti-B cell antibody (monoclonal) for the enumeration of T and B cells using fluorescent microscopy.